Genomic instability is a threat to cell survival and a major factor that drives tumorigenesis. During DNA replication, cells are particularly vulnerable to accumulate genomic instability as replication forks are prone to stall or collapse when encountering replication blocks or damaged DNA templates. To properly replicate the genome, cells rely on the replication checkpoint (RC), an evolutionary conserved signaling pathway that is constantly monitoring the integrity of DNA replication forks. Based on studies with clinical specimens, the RC has been proposed to constitute an early barrier against the progression of a number of cancers, including carcinomas of the lung, breast and colon. The phosphatidyl-inositol-3-kinase-like kinase ATR plays pivotal roles in the RC. In response to replication stress, ATR is rapidly activated at sites of damaged forks to initiate an elaborate signaling network that promotes fork stabilization and repair. Despite the importance, how ATR regulates the repair of replication-induced DNA lesions is not well understood. Important insights were revealed by our recent work in S. cerevisiae showing that Mec1 (yeast ATR) mediates the association of the replication factor Dpb11 (ortholog of human TopBP1) with Slx4, a scaffold protein that coordinates the action of DNA repair factors. While our work places Dpb11 and Slx4 at the heart of RC-mediated fork repair, how these proteins coordinate the action of repair pathways at damaged forks remains a wide open question. Furthermore, as the mammalian ortholog of Slx4 was just recently identified, how this highly conserved scaffold links RC-signaling to repair pathways emerges as a fundamental problem with implications for understanding genome maintenance and cancer. With the long-term goal of elucidating how RC-signaling maintains fork integrity, in Aim 1 we use yeast genetics as a powerful tool to define how the Mec1-Slx4-Dpb11 axis of RC-signaling controls repair pathways in response to replication blocks. In Aim 2, we use a new Slx4 gene-targeted mouse model to identify both conserved and potentially novel roles for mammalian Slx4 in repair pathways that prevent replication-induced genomic instability. We anticipate that these studies will establish Slx4 as a key RC-effector for replication fork repair in yeast and mammals. In Aim 3 we determine how Dpb11 controls the use of Slx4 and other repair effectors for lesion-specific DNA repair, including the repair of replication-induced double stranded breaks. The results will delineate how Slx4 functions in the RC and will unmask previously unappreciated roles for Dpb11 in repair pathways. Taken together, we expect that the work being proposed here will significantly enhance our understanding of how cells respond to replication stress. Given the direct relationship of RC-signaling with cancer, and the wide-spread use of replication stress as a strategy for cancer therapy, we expect our work to have broad implications for human health. PUBLIC HEALTH RELEVANCE: Our research will contribute to a better understanding of how cancer is initiated. The results could lead to more efficient strategies for cancer management by providing rationale for the development of highly specific drugs.